Several publications and patent documents are cited in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these citations is incorporated by reference herein.
Neurotrophic factors are considered to be vital for normal development of the nervous system. During development, neuronal target structures produce limited amounts of specific neurotrophic factors necessary for both the survival and differentiation of neurons projecting into the structures. The same factors have been found to be involved in the survival and/or maintenance of mature neurons.
A neurotrophic factor is defined as a substance capable of increasing and/or maintaining survival of a neuron population, and possibly affecting outgrowth of neurites (neuron processes) and certain other metabolic activities of a neuron. Neurotrophic factors are generally described as soluble molecules synthesized in the peripheral targets of neurons and transported to their cell bodies, where they exert their effects.
Studies with isolated neurotrophic factors have shown that exogenously added neurotrophic factors can exert their neurotrophic effects upon cultured neurons in vitro, or by administration to damaged or degenerated neurons in vivo. For this reason, various neurotrophic factors have received great attention as potential therapeutic agents for treatment of degenerative diseases of the central nervous system, as well as traumatic damage to the CNS. For example, nerve growth factor (NGF) has been shown to increase the survival, function and regeneration of cholinergic neurons in the basal forebrain. Degeneration of this population of cholinergic neurons has been associated with patients having Alzheimer's disease, and could be the primary neuronal defect responsible for the loss of cognitive function associated with Alzheimer's disease. NGF has been found to be synthesized and released from the target areas of these cholinergic neurons in the hippocampus and neurocortex, both areas of the brain associated with learning and memory. See Springer, J. E., Drug News and Perspectives, 4: 394-99 (1991). As another example, a dopaminergic neurotrophic factor (DNTF) has been purified and characterized, and found to promote survival and neurite outgrowth of dopaminergic neurons of the substantia nigra. DNTF is considered a potentially valuable therapeutic agent for the treatment of Parkinson's disease which involves degeneration of dopaminergic motor neurons of the central nervous system (U.S. Pat. No. 5,215,969 to Springer et al., 1993).
It can be seen from the foregoing examples that neurotrophic factors are a valuable source of therapeutic agents for the treatment of neuron damage and neurodegenerative disease. However, the development of such factors as therapeutic agents can be problematic. For example, it is difficult to determine the specificity of an endogenous neurotrophic agent, i.e., whether different factors exist for different nervous system pathways, and which neuron populations in those pathways are affected by a factor. In fact, many identified neurotrophic agents have been shown to have a wide range of biological functions, acting on both central and peripheral neurons, as well as non-neuronal cells in vitro (e.g., polypeptide growth factors and ciliary neurotrophic factor, CNTF). In the central nervous system, with its complex interconnections and heterogeneous neuron types, it is difficult to determine which neurotrophic factors are effective on a particular neuronal population. This difficulty is further exacerbated by the fact that many of the neurotrophic factors that have been characterized have been found to be closely related to one another. For example, it is now known that NGF possesses amino acid sequence homology to brain-derived neurotrophic factor (BNDF), a protein with similar, but not identical, in vitro properties as NGF (Barde et al., EMBO J., 1: 549-53, 1982; Leibrock et al., Nature, 341: 149-52, 1989). In fact, NGF, BNDF and the neurotrophin (NT) series have been classified as members of a superfamily of neurotrophic factors (NGF superfamily). Because of their similarity in amino acid sequence (and hence nucleotide sequences encoding the factor), it has been difficult to develop nucleic acid or antibody probes that are specific for a particular member of the family. The lack of a specific means for identifying a particular neurotrophic factor has hindered the elucidation of particular neuronal populations affected by a specific factor.
An additional obstacle to developing neurotrophic factors as therapeutic agents for treatment of damaged neurons is that few in vivo models exist to study the survival-promoting activity of these factors in the central nervous system. In order to develop a neurotrophic factor as an effective therapeutic agent for the treatment of neuron degeneration, it is important to be able to determine where in the central nervous system the neurotrophic factor operates, whether the treatment with exogenous neurotrophic factor is effective, and the concentration of neurotrophic factor effective for imparting a therapeutic effect. Such an objective would best be accomplished with a neurotrophic factor that is identifiable and distinct from other factors, that is capable of exerting an effect on many different neuron populations, and for which in vivo models are available to test the efficacy of the neurotrophic factor on a specific neuron population.
The neuron survival-promoting peptide Y-P30 was originally identified in the secretions of neural cells (neuroblastoma and retinoblastoma) subjected to oxidative stress (Cunningham, et al. 1998). Partially purified fractions of conditioned culture medium were screened in vitro until the active Y-P30 peptide was identified—the synthetic version of this peptide was then tested in vitro and in vivo and found to support neural cells which were degenerating for a variety of reasons, including oxidative stress and central nervous system trauma (Cunningham, et al. 1998; 2000). This peptide was later confirmed to be part of an endogenous human polypeptide (˜12 kiloDaltons) named DSEP after identification of the human cDNA encoding DSEP and the locus of the DSEP gene in human chromosomal region 12 q (Cunningham, et al. 2002). In that study, it was found that overexpression of the full length polypeptide in neural cells made them resistant to several forms of oxidative stress including that resulting from immune cell attack.
The contribution of inflammatory cells and their secretions to cell death after CNS injury or in neurodegenerative disorders is for the most part well established (Stoll, 1998). The principal immune cell participants in the response to traumatic CNS injury are monocyte derivatives (microglia/macrophages). These cells are the source of a number of inflammatory agents that may contribute to neuron death, including superoxide anion, nitric oxide, IL-1β, and TNFα (reviewed by Rothwell, et al 1996, Stoll et al 1998, Jander, et al 1998, 2000; and Turrin, et al 2001). TNFα is best known for its cytotoxic activity outside the nervous system, but also has pronounced toxic activity on neural cells after brain injury (Barone, et al, 1997; Lavine, et al 1998). Both overexpression of the full length DSEP molecule and application of Y-P30 inhibits the appearance and differentiation of macrophages and microglia (Cunningham et al. 1998, 2000, 2002).
Steriod anti-inflammatory drugs currently used to treat nervous system injury and other disorders with an inflammatory component operate in part by stimulating the production of endogenous inhibitors of phospholipases (A2) (PLA2) which are the enzymes responsible for the production of several lipid mediators of inflammation (Flower, R J et al. 1979). PLA2 enzymes and downstream participants in this pathway play a role in chronic neurodegenerative disorders including Alzheimer's disease (Farooqui A A, et al., 1999; Hull M, et al., 2002).
Therefore, a need exists in the art for identification and testing in vivo of new neurotrophic factors which are distinct from other factors, exert an effect on many different neurons, and/or which can act as PLA2 inhibitors, to facilitate the development of new therapies for neurodegenerative disorders and for other diseases with an inflammatory component.